Class-specific restrictions define primase interactions with DNA template and replicative helicase

Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases. Key residues in the β4 strand of the primase zinc-binding domain defined class-associated trinucleotide recognition and substitution of these amino acids transferred specificity across classes. A change in template recognition provided functional evidence for interaction in trans between the zinc-binding domain and RNA polymerase domain of two separate primases. Helicase binding to the primase C-terminal helicase-binding domain modulated RNA primer length in a species-specific manner and productive interactions paralleled genetic relatedness. Results demonstrated that primase template specificity is conserved within a bacterial class, whereas the primase–helicase interaction has co-evolved within each species

Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase.

The clamp-loader complex plays a crucial role in DNA replication by loading the β-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ’ Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model E. coli. Based on this information a homology model of the Bacillus subtilis τ3-δ-δ' complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ' binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared to the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg2+ conformational changes in DnaB and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilise its interaction with τ3

<em>E. coli</em> Fis Protein Insulates the <em>cbpA</em> Gene from Uncontrolled Transcription

<div><p>The <em>Escherichia coli</em> curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and co-chaperone. It is expressed at high levels as cells enter stationary phase. Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. We show that Fis, the dominant growth-phase nucleoid protein, prevents CbpA expression in growing cells. Regulation by Fis involves an unusual “insulation” mechanism. Thus, Fis protects <em>cbpA</em> from the effects of a distal promoter, located in an adjacent gene. In stationary phase, when Fis levels are low, CbpA binds the <em>E. coli</em> chromosome with a preference for the intrinsically curved Ter macrodomain. Disruption of the <em>cbpA</em> gene prompts dramatic changes in DNA topology. Thus, our work identifies a novel role for Fis and incorporates CbpA into the growing network of factors that mediate bacterial chromosome structure.</p> </div

Repression of transcription from the P4 and P6 promoters by Fis.

<p>(A) Repression by Fis <i>in vitro</i>. i) Schematic of the <i>cbpA</i> regulatory region DNA used for <i>in vitro</i> transcription assays. Promoters are shown by coloured arrows and the λ<i>oop</i> transcription terminator is shown by a black “lollipop”. The Fis binding element is shown as a yellow bar. Different mRNA transcripts are shown by coloured wavy lines. ii) mRNA transcripts generated by a combination of σ⁷⁰ and σ³⁸ associated RNA polymerase (400 nM each) in the presence and absence of Fis (500 nM). Transcripts are labelled according to the scheme in panel i). (B) Repression by Fis <i>in vivo</i> requires P6 and the Fis binding element. Different <i>cbpA</i>::<i>lacZ</i> fusions are illustrated. Promoters are shown by coloured arrows. The Fis binding element is shown as a yellow bar. LacZ activity values from growing JCB387 and JCB3871Δ<i>fis</i> cells are given adjacent to each promoter::<i>lacZ</i> fusion. The fold repression by Fis is shown in parenthesis.</p

CbpA expression is uncoupled from growth-phase in a <i>fis</i> mutant.

<p>(A) Activity of a <i>cbpA</i>::<i>lacZ</i> fusion in different growth-phases. i) Schematic representation of the <i>cbpA</i>::<i>lacZ</i> fusion. A 302 base pair DNA fragment, encompassing the <i>cbpA</i> start codon, the entire <i>cbpA</i>-<i>yccE</i> intergenic region, and a portion of the <i>yccE</i> gene, was cloned upstream of <i>lacZ</i> in the low copy number <i>lacZ</i> reporter plasmid pRW50. ii) The graph shows LacZ activity calculated for either wild type or Δ<i>fis</i> cells carrying the plasmid construct shown in panel i). iii) Western blot showing Fis levels in <i>E. coli</i> K-12 throughout growth and in overnight cultures. The blots were also probed with antibodies against RpoA as a control. (B) Fis has a high affinity for the <i>cbpA</i> regulatory region. The graph illustrates the binding of Fis to different DNA fragments (see key). The raw EMSA data are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003152#pgen.1003152.s001" target="_blank">Figure S1</a>.</p

Chromosome-wide distribution of CbpA in starved <i>E. coli</i>.

<p>(A) Distribution of CbpA across the <i>E. coli</i> chromosome. i) Genome-wide view of CbpA binding in starved MC108 cells. The figure shows ChIP-chip data for CbpA binding plotted against features of the <i>E. coli</i> genome in the form of a genome atlas. The data have been averaged across a 100,000 base pair window. The four chromosomal macrodomains (MD) are labelled ii) CbpA ChIP-chip data for a small section of the <i>E. coli</i> chromosome. The data have been averaged across a 10,000 base pair window. (B) Relationship between CbpA binding and DNA GC content. The graph shows the average CbpA binding signal plotted against the GC content of probes on the DNA microarray. The average GC content of the <i>E. coli</i> K-12 chromosome is shown by a dashed line. Very GC rich and GC poor probes were excluded during microarray design and are thus absent. (C) Effect of CbpA on DNA supercoiling <i>in vivo</i>. Panel i) shows an image of a 1% (v/v) agarose gel containing 2.5 µg/ml chloroquine. Plasmids were isolated from different genetic backgrounds and different stages of growth as indicated. Panel ii) also shows an image of a 1% (v/v) agarose gel containing 2.5 µg/ml chloroquine. Plasmids were isolated from rapidly dividing cells.</p

The nucleoid protein response to starvation in <i>E. coli</i>.

<p>(A) Model of <i>cbpA</i> regulation by Fis. (B) Model for staged induction of <i>cbpA</i> and <i>dps</i>.</p